Naomi KASHIWAZAKI, Ph' D'

1
Reproductive Bioscience and Biotechnology
-Basics, Application and Venture Business-
Application of Reproductive Bioscience and
Biotechnology Biotechnology in laboratory
animals"
Naomi KASHIWAZAKI, Ph. D. AZABU UNIVERSITY
2
Reproductive technologies are extensively used as
tools for transgenesis and banking genetic
resources etc in laboratory animal sciences. The
following topics are focused in this lecture.
Generation of transgenic animals
Cryopreservation of spermatozoa
Cryopreservation of oocytes/embryos
Laboratory of Animal Reproduction, Azabu
University
3
What is transgenic animals?
Laboratory of Animal Reproduction, Azabu
University
4
Nuclei
Whole body
Cells
Chromosomes
Transgene
Transgenic animals Gene transfer to genome (germ
line)
DNA
Laboratory of Animal Reproduction, Azabu
University
5
V
V
Transgenic
Wild type (Non-transgenic)
Function phenotype
Transgenic animals
Laboratory of Animal Reproduction, Azabu
University
6
Why make transgenic animals?
Laboratory of Animal Reproduction, Azabu
University
7
Why make transgenic animals?

• To gain new knowledge (powerful research tool)
• To decipher the genetic code
• To study the genetic control of physiological
  system
• To build genetic models
• To improve animal production
• To produce animal products

Laboratory of Animal Reproduction, Azabu
University
8
Transgenesis

• DNA microinjection method
• Embryonic stem cells method (Targeting)
• Virus mediated method
• Sperm vector method
• Somatic cell clone method

Laboratory of Animal Reproduction, Azabu
University
9
DNA microinjection in rats
10
DNA microinjection in pigs
11
0.1 mm (100 ?m)
Porcine 1- 2-cell stage embryos
12
DNA microinjection in pigs
13
An inverted microscopy attached macromanipulators
14
Transfer of DNA injected embryos in pigs
15
Newborn piglets derived from DNA injected embryos
16
12 88 PC NP

B

B
36 7 12 88 PC NP

A

A
S
CN/hGH)
S
CN/hGH)
1
(a
1
(a
36 7
36 7
C
C
PCR Analysis of the Tg Pigs
PCR Analysis of the Tg Pigs
A
A


GH 208


using by the primer GH 106
and
using by the primer GH 106
and


B
B

BAC 004


GH102
using by the primer
and
using by the primer

C
C

BBCG 06


using by the primer
and
BBCG 16
using by the primer

CN/hGH)
CN/hGH)
(b
(b
17
Production of transgenic pigs by DNA
microinjection
Total
Transgene
b
S1CN-hGH
b
CN-hGH
a
b
of injected/transferred embryos
305
287
592
of Tg
2
4
2
of integration
0.7
0.7
0.7
of Tg expression
1
0
1
0
0.3
0.2
of Tg expression
of treansferred recipients
12
11
23
of pregnant recipients
7
7
14
7

of farrowed recipients
5
12
58.3
of farrowing
41.7
52.2
of offspring
41
29
70
Survival ()

13.4
10.1
11.8
18
Growth hormone
Milk protein (casein)
Promoter
Structure gene
Fused gene transgene
19
Transgenic rats carrying the EGFP gene
20
Milking from the transgenic sow
21
Transgenic via DNA microinjection
1. Random integration of transgene
2. Low integration of transgene
3. Reliable method
Laboratory of Animal Reproduction, Azabu
University
22
Transgenesis

• DNA microinjection method
• Embryonic stem cells method (Targeting)
• Virus mediated method
• Sperm vector method
• Somatic cell clone method

Laboratory of Animal Reproduction, Azabu
University
23
Transgene
ES cells
Selection
Mice
Blastocysts
Transgenesis via ES cells (1)
24
Blastocyst injection
Selected (Tg) cells

Natural mating
Transgenic
Chimeras
Embryo transfer
Transgenesis via ES cells (2)
25
ES-like cells of the pig
26
Blastocyst injection in pigs
27
Cell-injection to morula in pigs
28
Chimeric (Large White - Duroc) pig generated by
blastocyst injection
29
Transgenesis via ES cells
1. Gene knock-out (targeting)
2. ES cells that can transmit to germ line
is only available in mice
30
Gene targeting is required in other species of
mice!
31
Transgenesis

• DNA microinjection method
• Embryonic stem cells method (Targeting)
• Virus mediated method
• Sperm vector method
• Somatic cell clone method

Laboratory of Animal Reproduction, Azabu
University
32
Making transgenic clones multistep procedures

• Selection of cultured somatic cells (Tg cells)
• as nuclear donor
• Enucleation
• Nuclear transfer
• Artficial activation
• Embryo transfer

Laboratory of Animal Reproduction, Azabu
University
33
VVVVV
VVVVV
VVVVV
VVVVV
Enucleation
34
Selection
Culture of somatic cells
Artificial activation
Nuclear trabsfer
Nuclear transfer and artificial activation
35
Reconstructed embryos
Transgenic clones
Embryo transfer
Embryo transfer of the reconstructed embryos
36
Selected cells
Transgenic clones
Embryo transfer
Transgenesis via nuclear transfer
37
Problems of transgenic clones

• Low efficiency
• Malformation

Laboratory of Animal Reproduction, Azabu
University
38
Experimental farm for producing transgenic pigs
39
Surgery of embryo recovery and transfer
40
Showing before surgery
41
Anethsia
42
Transport to the table (1)
43
Transport to the table (2)
44
Surgical table for pigs
45
Inhalation anethesia apparatus
46
Reproductive organs (ovary, oviduct, Uterus) of
the pig
47
Embryo flushing in pigs
48
Embryo transfer to the oviduct in pigs
49
In vitro production (IVP) of porcine embryos
50
Piglets derived from IVM/IVF/IVC (IVP) embryos
51
Generation of transgenic animals
Cryopreservation of spermatozoa
Cryopreservation of oocytes/embryos
Laboratory of Animal Reproduction, Azabu
University
52
Why cryopreserve spermatoza and
oocytes/embryos?
Laboratory of Animal Reproduction, Azabu
University
53
Why cryopreserve spermatoza and
oocytes/embryos?

• Genetic resources including transgenic
• animals can be banked effectively.

• Banked genetic resources can be
• distributed effectively and safely.

Laboratory of Animal Reproduction, Azabu
University
54
Rat male (SD)
Epididymedes
Collection of epididymal spermatozoa
55
First egg-yolk diluent
Dissection
Second egg-yolk diluent
At 5?
15? 30 min
5? 30 min
56
Loading into 0.25 ml straws
The straws were exposed to vapor of liquid
nitrogen (LN) and plunged into LN.
?????
57
Thawing of cryopreserved straws
58
Effect of glycerol and 0.7 Equex Stem in
freezing medium on post-thaw sperm motility and
plasma membrane integrity in the rat
-
-

• Means SEM, a vs. b Plt0.05, c vs. d Plt0.05,
  c vs. e Plt 0.01, f vs. g Plt0.05

59
Results of Intrauterine Inseminasion with
Cryopreserved Rat Epididimal Spermatozoa
Litter size
of
Delivery
Pregnancy
Inseminated
Sperm
in Pregnant
Sperm
()
()
Females
Motility
SD 9.61.5 4/5 (80) 3/5 (60)
3.7
Wistar 10.00.0 4/5 (80) 3/5
(60) 4.3
Long-Evans 5.73.3 4/5 (80) 3/5 (60)
2.0
WKY/NCrj 10.76.7 5/5 (100) 2/5 (40) 4.0
BN/Crj 2.00.6 4/5 (80) 3/5 (60)
3.0
F344/DuCrj 10.02.5 5/5 (100) 4/5 (80)
7.0
LEW/Crj 6.71.7 3/5 (60) 1/5 (20)
4.0
GK/Crjb 15.63.3 1/2 (50) 1/2 (50)
3.0
WTC. ZI-zi 8.71.8 4/5 (80) 4/5 (80)
6.7
TRM 9.33.3 5/5 (100) 5/5
(100) 4.8
EGFP/Tg 12.31.5 4/5 (80) 3/5 (60)
6.7
A/Tg 10.62.0 5/5 (100) 5/5
(100) 6.0
60
Semen collection in rabbits
61
Froze/thawed semen
Artificial insemination in rabbits
62
Post-thaw sperm motility frozen with 2 acetamide
Laboratory of Animal Reproduction, Azabu
University
63
Artificial insemination with semen frozen with 2
glycerol or acetamide
Semen Kindling
Offspring 2.0 acetamide
7/8 ( 87.5 ) 5.33.3 2.0
glycerol 4/8 ( 50.0 )
1.51.7
Plt0.05
64
Generation of transgenic animals
Cryopreservation of spermatozoa
Cryopreservation of oocytes/embryos
Laboratory of Animal Reproduction, Azabu
University
65
Cryopreservation of oocytes/embryos
Vitrification High survival, No ice formation,
No pragrammable freezer, Quick treatment, High
CPA(s) Conventional slow freezing Icee
formation, Low CPA(s)
Laboratory of Animal Reproduction, Azabu
University
66
Reproductive technologies are extensively used as
tools for transgenesis and banking genetic
resources etc in laboratory animal sciences.
Generation of transgenic animals
Cryopreservation of spermatozoa and
oocytes/embryos Related technologies are in
vitro fertilization, intracytoplasmic sperm
injection, artificial insemination, embryo
culture, embryo transfer.
Laboratory of Animal Reproduction, Azabu
University
67

• Never forget careful observation,
• managemen and ethics of laboratory animals!
• Keep on studying, and keep on smiling!!

Laboratory of Animal Reproduction, Azabu
University